Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: The 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA

ANALYTICAL

BIOCHEMISTRY

129, I-13

(1983)

Complete Analysis of tRN&Modified Nucleosides by High-Performance Liquid Chromatography: The 29 Modified Nucleosides of Salmonella typhimurium and Escherichia co/i tRNA MARTIN Department

BUCK, of Biochemistry,

MEGAN

CONNICK,

University

AND BRUCE N. AMES

of California,

Berkeley,

California

94720

Received August 9, 1982 A high-performance liquid chromatography (HPLC) method has been developed to quantify the major and modified nucleoside composition of total, unfractionated transfer RNA. The method is rapid and sensitive and offers a high degree of chromatographic resolution suitable for quantifying both stable and unstable modified nucleosides. It is nondestructive and allows the recovery of nucleosides for further characterization. We apply the method in the analysis of the 29 modified nucleosides in tRNA from Salmonella typhimurium (and Escherichia co/i) and show it to be useful in examining changes in the modified nucleoside content of tRNA. Such changes may be important in regulation.

contains a large number of modified nucleosides, several of which may play an important role in cellular regulation (1). The modifications are extremely diverse, ranging from simple methylations to complex hypermodifications requiring several enzyme activities in their formation (1). Certain modifications are extremely rare, occurring in only one or two of the tRNA species found in total unfractionated tRNA. Therefore, base analysis of tRNA requires a high-resolution method which is sensitive and does not introduce artifacts due to the inherent instability of several of the modified nucleosides. Sensitivity can be achieved by postlabeling techniques using either the trialcohol procedure of Randerath (2) or the 5’ postlabeling of 3’mononucleotides using [T-~~P]ATP and T4 polynucleotide kinase (3,4). However, the trialcohol procedure is limited to base-modified nucleosides and some modified nucleosides, in particular thiolated nucleosides, are destroyed by this method. In addition, certain modified nucleotides are less susceptible to phosphorylation with T4 polynucleotide kinase than others and a quantitative estimation of nucleotide composition may not al-

ways be possible with this method (3). Thus, although both techniques are very useful and are in widespread use, they have limitations. Therefore, the high-resolution thin-layer chromatography systems available for separating modified nucleosides and nucleotides are limited by the method chosen to detect the modified nucleoside (5- 10). Localization of nucleosides on thin-layer chromatography plates by uv absorption requires relatively large amounts of material, and the radioactive labeling of the nucleoside or nucleotide to enhance the level of its detection is subject to in viva and in vitro constraints. Not all sources of tRNA are suited to in vivo labeling procedures yielding uniformly radioactive tRNA, and in vitro postlabeling techniques have certain inherent limitations mentioned above. In this paper we describe the application of reverse-phase HPLC to the analysis of modified nucleosides from tRNA. HPLC procedures and methods have been developed and applied to this field before (11,12) and have been used in tRNA base composition studies ( 13- 15) but were not ideal for our analysis of the role of modified nucleosides in regulation. We have thus paid particular attention to a

tRNA

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0003-2697/83/030001-13$03.00/O Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

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method of tRNA isolation and digestion compatible with HPLC analysis and optimizing the separation of all of the 29 modified nucleosides present in Salmonella typhimurium tRNA using a rapid gradient elution procedure. The method we describe is applicable to examining in viva alterations in the modified nucleoside content of tRNA which may be of regulatory significance to the cell. A detailed analysis of the modified nucleoside composition of S. typhimurium tRNA is presented. METHODS

Bacterial strains. Wild type S. typhimurium LT2 was used throughout this study. Media. Minimal salts N-C- media ( 16) were

supplemented with 0.4% (w/v) glucose and 10 NH&l as carbon and nitrogen sources, respectively. Culture methods. Inocula (20 ml) were grown overnight at 37°C without shaking in minimal media containing 0.04% glucose. These cells were diluted into 200 ml of fresh medium containing 0.4% glucose to give an initial absorbance at 650 nm of 0.02. Cells were grown with shaking (250 rpm) in a lliter flask until the Ah50 reached 1.0 (8 X lo* cells/ml). Growth was followed spectrophotometrically with a Zeiss Model PMQ 11 spectrophotometer. Preparation of tRNA. Bacteria from a 200ml culture were quickly chilled on ice and collected by centrifugation at 4°C. The cells were then washed once with cold 0.01 M Mg acetate, 0.05 M Na acetate, 0.15 M NaCl, pH 4.5, and resuspended in 5-7 ml of the same buffer. The cell suspension was then shaken with an equal volume of 80% (w/v) redistilled phenol/water for 20 min in the cold. The emulsion was broken by centrifugation and the resultant phenol phase reextracted with 2-3 ml of pH 4.5 buffer. The aqueous phases were pooled and 0.1 vol of 20% KAc, pH 4.5, was added. The aqueous phase was then made 2 M in LiCl by the addition of 12 M LiCl. After a period of 4 h on ice, to allow highmolecular-weight RNA to precipitate ( 17), the tnM

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aqueous phase was centrifuged (20 min, 5OOOg) and the RNA in the supernatant precipitated by the addition of 3 vol of ethanol. The precipitate was dissolved in 1 ml of 0.1 M Tris-HCl, 0.01 M MgClz, pH 7.5, and applied to a 2.5 X 1.O-cm column of DEAE-cellulose followed by 2 ml of the same buffer and 7 ml of Tris-Mg buffer (pH 7.5) containing 0.2 M NaCl. Transfer RNA was eluted with 10 ml of Tris-Mg buffer containing 1.O M NaCl and recovered by ethanol precipitation. The 2 M LiCl salt fractionation was then repeated in a small volume (150-200 ~1) to ensure efficient removal of rRNA, and the tRNA was then precipitated with ethanol. The final tRNA precipitate was washed once with 80% ethanol to remove salt prior to digestion. Enzymatic hydrolysis of tRNA. Transfer RNA (20 AZ@ units/ml) was digested in 0.08 M ammonium formate buffer, pH 7.6,0.5 mM MgC12 containing per milliliter: 0.5 mg snake venom phosphodiesterase (Worthington), 0.5 mg ribonuclease A (Sigma, Type 11 I-R), and 0.5 mg bacterial alkaline phosphatase (Sigma, Type 111-R). Digestion was carried out for 18-20 h at 37°C after which 25 units of ribonuclease T, and 12.5 units of ribonuclease T2 were added per milliliter and the incubation continued for a further 2 h. Ribonuclease T2 digestion of tRNA was carried out in 0.025 M ammonium acetate, pH 4.5, 1 IIIM EDTA containing 20 Azm/ml tRNA, and 5 units of ribonuclease Tz per A260 unit of tRNA. After 15-16 h incubation at 37”C, the digest was lyophilized to dryness and resuspended in 0.08 M ammonium formate, pH 7.6, 1 mM MgC12 containing 0.5 mg/ml bacterial alkaline phosphatase and equal in volume to the original ribonuclease T2 digestion volume. The incubation was then continued for a further 5-6 h. Preparation of nucleosidesfor HPLC. Nu-

cleosides from a 100-~1 digest were mixed with 20 mg of wet, packed silicic acid ( 100 mesh) to remove protein prior to chromatography. After gentle mixing on ice for 15 min the silicit acid was removed by centrifugation and the nucleosides were stored at -20°C. Im-

COMPLETE

ANALYSIS

OF tRNA-MODIFIED

mediately before HPLC analysis, the nucleoside solution was filtered through a 0.45~pm cellulose acetate filter (Bioanalytical Systems centrifugal microfilter) to remove traces of particulate silicic acid. The recovery volume was greater than 95%. Boronate afinity chromatography. A tRNA hydrolysate equivalent to 2-3 AZ60 units of tRNA was adjusted to pH 8.8 by the addition of an equal volume of 2.0 M ammonium acetate, pH 8.8, and applied to 0.7 X 2.0-cm column of Bio-Rad Affi-Gel60 1. The column was washed with 5 ml of 1.0 M ammonium acetate, pH 8.8, and the flow through collected. The bound nucleosides were eluted with 7 ml of 0.1 M acetic acid. Both eluates were lyophilized to dryness and each was resuspended in a volume of water equivalent to the original volume of hydrolysate applied to the column. Synthesis of mcmo*U.’ Acid methanolysis of 1 mg of cmo’U was carried out in 100 ~1 of anhydrous methanol containing 0.8 ~1 concentrated HCl for 20 h at 37°C. The reaction mix was neutralized by the addition of 100 ~1 0.25 M ammonium acetate, pH 6.0, and ’ Abbreviations used: Am, 2’-O-methyladenosine; Gm, 2’-O-methylguanosine; Urn, 2’-O-methyluridine; Cm, 2’-O-methylcytidine; m6A, 6-methyladenosine; m*A, 2-methyladenosine; m’A, I-methyladenosine; i6A, p-isopentenyladenosine; c-io6A, cis-ribosylzeatin; t-io6A, transribosylzeatin; ms2i6A, 2-methylthio-iV6-isopentenyladenosine; cis-ms2i06A, cis-2-methylthioribosyzeatin; ms*A, 2-methylthioadenosine; @A, N-[9-@-ribofuranosylpurin-6-yl)carbamoyl]threonine; mt6A, N-[9-p-D-ribofuranosylpurin-6-yl)N-methylcarbamoyllthreonine; m@‘A, 2 - methylthio -N - [9-p - D - ribofuranosylpurin - 6 - yl) carbamoyllthreonine; m6Am, 2’-O-methyl-6-methyladenosine: m:A, N”dimethyladenosine; I. inosine: m’l, 1-methylinosine; m’G, 7-methylguanosine: m*G, 2methylguanosine; m’G. 1-methylguanosine: m:G, N*-dimethylguanosine; Q. queuosine; Y, wybutosine; #, pseudouridine; mSU, 5-methyluridine (ribothymidine); m3U, 3-methyluridine; s4U, 4-thiouridine; m3s2U, 5-methyl-2thiouridine; mnm5s2U, 5-methylaminomethyl-2-thiouridine; cmnm?.*U, 5-carboxymethylaminomethyl-2thiouridine; mcm5s2U, 5-(methoxycarbonylmethyl)-2thiouridine; cmo%J, uridine 5-oxyacetic acid; mcmo%J, methyl ester of cmo’U; acp’U, 3-(3-amino-3-carboxypropyl)uridine; m5C, 5-methylcytidine; szC, 2-thiocytidine; a&, N4-acetylcytidine; dN, deoxynucleoside; 2’,3’-cNMP, 2,3’-cyclic mononucleotide.

NUCLEOSIDES

3

concentrated KOH and then lyophilized to dryness. The product of the reaction moved with an Rf of 0.83 in ethanol/l M ammonium acetate, pH 7.5 (7/3 v/v) on microcrystalline cellulose thin-layer chromatography plates (Polygram ccl 400) and was converted to the starting material (Rf 0.28) following alkaline hydrolysis (0.1 M KOH, 37°C for 18 h). Additionally, the product gave one major peak on HPLC which was alkali labile and eluted later than cmo’U. These characteristics are consistent with the methanolysis product being mcmo’U (18). HPLC methodology. All HPLC buffers were prepared in Milli-Q water (Millipore water purification system). Buffer A, 0.25 M ammonium acetate, pH 6.0, was purified by passage over a 25 X l-cm preparative 5-pm C18 reverse-phase column. Buffer B was 40/60 (v/v) acetonitrile/water. Both buffers were stored at 4”C, filtered through a 0.45-pm filter, and degassed by purging with high-purity helium prior to use. Nucleosides from 1 AZ60 unit of bulk unfractionated tRNA or approximately 0.2 A260unit of pure isoacceptor tRNA in a volume of 50 ~1 were injected (Waters automated injection module 710B) and resolved on a 250 X 4.6-mm Supelco 5-pm C18 reverse-phase analytical column. Chromatography was carried out at ambient temperature with a flow rate of 2 ml/min and a gradient of 100% A to 100% B as detailed below. The solvent delivery system consisted of two Waters M6000A pumps. Absorbance at 254 and 280 nm was monitored with a Waters 440 dual-wavelength absorbance detector. Absorption data were integrated using a Spectra Physics SP4020 data interface and SP4000 central processor. The elution gradient was generated with a Waters Model 720 system controller prior to linearization of the pump control.* In the following description of the gradient, the first number refers to the percentage buffer B being pumped and the sec* Linearization of the Model 720 systemcontroller generates an effectively shallower gradient in the early region of the HPLC run with increased retention times for compounds eluting near guanosine.

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RESULTS

ond number to the elapsed time in minutes: 0, 0; 0, 3; 5, 10; 25, 25; 50, 30; 75, 34; 75,

37; 100, 45; and 100, 48. All increases in B were linear (gradient 6) with the exception of the 0 to 5% increase which was concave (gradient 7). The gradient is presented schematically in Fig. 2. Quantljication of nucleosides. Nucleosides were dissolved in 0.01 M phosphate buffer at the appropriate pH and their exact concentrations determined spectrophotometrically at X max from the published molar extinction coefficients ( 19). The ms2i6A and ms2i06A were prepared in ethanol for spectrophotometric standardization, lyophilized, and then dissolved in a known volume of water before HPLC. Data for the integrated absorbance of mcmo’U at 254 nm under HPLC conditions were calculated from the yield of cmo5U following alkaline hydrolysis of mcmo’U and HPLC separation of the hydrolysis products. Each standardized nucleoside solution was chromatographed under HPLC conditions and the integrated absorbance at 254 and 280 nm determined for the known amount of nucleoside injected. This integration value was used to determine the molar concentration of each nucleoside in tRNA hydrolysates. Integration was shown to be linear over the range of nucleoside concentrations found in tRNA by dilution of both a standard mix of nucleosides and a tRNA hydrolysate. Source of modiJied nucleosides. Nucleosides t6A, mt6A, ms2t6A, t-io6A, c-io6A, cms2i06A, OH-i6A,3 ms2i6A, ms2A, Q, acp3U, cmo’U, m5s2U, mnm5s2U, mcm5s2U, and s2C were kind gifts from G. Chheda, H. Ishikura, N. Leonard, M. Lipsett, B. McLennan, J. McCloskey, S. Nishimura, B. Void, and H. Vorbruggen. All other modified nucleosides were obtained from P-L Biochemicals, Sigma Chemical Company, or Vega-Fox Biochemical Company. E. coli tRNAG’” and tRNATy’ were obtained from Boehringer-Mannheim, and yeast tRNAPhe was obtained from Sigma. 3 Markovnikov of the isopentenyl

addition of water across the double bond side chain of i6A.

Preparation

of tRNA

The combined use of lithium chloride fractionation and DEAE-cellulose chromatography was found to be the most convenient and effective method of preparing bulk S. typhimurium tRNA free of rRNA and DNA. Both rRNA and DNA contain modified nucleosides in addition to those found in tRNA hydrolysates (20). Additionally, rRNA contains several modified nucleosides found in tRNA (2 l), and its presence therefore interferes with the quantitation of these nucleosides if the level of rRNA contamination is uncertain. Before treatment with lithium chloride, relatively high levels of m2G, m:G, and m6A modified nucleosides known to be present in E. coli rRNA (2 1), and the deoxy nucleosides are present in the hydrolysate of crude tRNA. Following LiCl fractionation, the levels of these nucleosides are substantially reduced, and mcmo’U previously masked by dA is then detected (see Fig. 3). Complete removal of high-molecular-weight rRNA and DNA was only achieved using both DEAE-cellulose chromatography and lithium chloride fractionation. tRNA prepared in this manner was comparable to polyacrylamide gel-purified tRNA in nucleoside composition, indicating the absence of rRNA. Hydrolysis

of tRNA

Two methods of tRNA hydrolysis were employed, one using a single-step digestion with snake venom phosphodiesterase, pancreatic ribonuclease, and alkaline phosphatase and the other employing ribonuclease T2 digestion at pH 4.5 followed by alkaline phosphatase treatment at pH 7.6. Although the single-step digestion procedure has been evaluated before (5), it was found necessary to raise the enzyme concentration to achieve complete hydrolysis of the tRNA. This is attributed in part to the higher concentration of tRNA in the digestion than in the published procedure (5). Using low enzyme concentrations resulted in the ac-

COMPLETE

ANALYSIS

OF

tRNA-MODIFIED

NUCLEOSIDES

5

cumulation of the dinucleotide GmG and Analysis of S. typhimurium tRNA by HPLC several other unidentified peaks in the HPLC The retention times and A2,,/A2, absorprofile which eluted between s4U and Q, A, bance ratios of modified nucleosides comand m6A. These are assumed to be limited digestion products, several of which are found monly found in tRNA are shown in Fig. 2. in ribonuclease Tz digests, indicating that they To identify each uv-absorbing compound in accumulate as a result of poor cleavage ad- S. typhimurium tRNA hydrolysates which was jacent to ribose methylated nucleosides. The resolved by HPLC, cochromatography experretention time of the dinucleotide GmG was iments were performed with authentic nudetermined from the ribonuclease T2 digest cleosides. Every nucleoside in S. typhimurium of tRNATy’ (Fig. 1C). The release of hyper- tRNA was identified on the basis of its specmodified nucleosides ($‘A, mt’A, ms2i06A, tral ratio and coelution with a known stanms2i6A) was not as severely affected by low dard. In addition, the tRNA digest was subenzyme levels. We selected a procedure in jected to boronate affinity chromatography to which ribonucleases T2 and T, were included examine the possible presence of nonnucleoin the digestion to remove 2’,3’-cyclic nucleoside uv-absorbing material and to confirm the tides which were detected at low levels in the identity of the ribose-methylated nucleosides. tRNA hydrolysate and which elute close to This procedure allows the separation of small molecules with cis-diol functionality (includseveral of the modified nucleosides (Fig. 2). The modified nucleoside composition of ing all nucleosides except those with ribose yeast tRNAPh” was determined using HPLC methylations) from those lacking this functo illustrate the complete release of the ribose- tion (24, 25). The nonbinding fraction from methylated nucleosides Cm and Gm under boronate chromatography was found to conthe digestion conditions chosen. The result is tain only the ribose-methylated nucleosides shown in Fig. 1A. There was very little con- and small amounts of each of the major nuversion of m’A to m6A during the digestion cleosides. Treatment of the nonbinding fmc(22), and all nucleosides were obtained in the tion with ribonuclease T2 and alkaline phosmolar yields expected from the published phatase showed it to be free of any 2, 3’-cyclic tRNA sequence (23). This included m’G nucleotides or 3’-monophosphates. which is known to be alkali labile (2 1). DigesThe nucleoside composition analysis data tion of E. coli tRNATy’ and tRNAG”’ (Figs. for unfractionated S. typhimurium tRNA are 1B, C, D) also yielded nucleosides in amounts shown in Fig. 3 and the accompanying Table predicted from published data (23). The in1. All 24 modified nucleosides reported to be tegrated absorption data at 254 nm for nu- present in E. coli and S. typhimurium tRNA cleosides Q and mnm5s2U, derived from di- were detected and quantified (21, 26, 27). gests of tRNATy’ and tRNAG’“, was used to HPLC analysis of tRNA hydrolysates from six estimate the levels of these nucleosides in S. E. coli strains (28) has shown them to be very typhimurium tRNA. As shown by the exper- similar in composition to S. typhimurium imental nucleoside composition data for the tRNA. The presence of ms2i06A in S. typhitRNAPh”, tRNATy’, and tRNAG’“, treatment murium tRNA (and its absence from E. coli of the tRNA digest with silicic acid to remove tRNA) has been confirmed by a variety of protein does not result in the loss of any nu- analytical methods (28). U* (Fig. 3) has been cleosides. The addition of up to 60 mg of si- tentatively identified as cmnm5s2U (28). In licit acid to S. typhimurium tRNA hydrolyaddition to cmnm5s2U and ms2i06A three sates or standard nucleoside mixes did not peaks of uv-absorbing material, designated Xi, cause the preferential loss of any known nu- X2, and X3, were detected in S. typhimurium cleoside. Such treatment resulted in only a tRNA which did not correspond to any of the slight dilution of the hydrolysate. standard nucleosides (Fig. 2) and remain un-

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PIG. 1. HPLC elution profiles of digests of purified tRNAs. Approximately 0.2-0.3 AzM) units of tRNA was analyzed. The absorbance at 254 nm was plotted. (A) Yeast tRNA”‘; (B) E. cdi tRNATW; (C) ribonuckase T2 digest of E. coIi tRNATF; (D) E. co/i tRNAG’“. (i) Experimentally determined nucleoside composition expressed as residues per tRNA chain. Data were normalized to a pseudouridine content of 2 residues per chain; (ii) standard deviation; (iii) nucleoside composition from published sequences (23). The level of m6A in the tRNA phedigest was determined by chromatographing 1 A260 unit of hydrolysate 6

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and found to be 0.4 mo1/2 mol $. Although a standard of Y was not available for comparison, the peak designated Y is assigned on the basis of its hydrophobicity and fluorescent nature. Fluorescence emission was monitored with a Schoeffel Model FS970 fluorometer at 340 nm using a 248-nm excitation wavelength (25). The numbers in parentheses (D) refer to the nucleoside composition determined from a ribonuclease T2 digest of tRNA”‘“. Values for Q, GmG, and mnm%*LJ are expressed relative to a mean value determined from ribonuclease T2 and the single-step digestion procedures.

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Time FIG. 2. Chromatographic separation pound are absorbance ratios (254/280

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of nucleosides and digestion products. Numbers nm). The elution gradient is shown in the lower

characterized. These compounds were also detected in ribonuclease T2 digests and were shown to be boronate binding, indicating they are unidentified nucleosides (see below). Although the HPLC separation of the nucleosides provides a high degree of resolution, the region containing nucleosides Q and Gm is dominated by the high level of Gm normally found in S. typhimurium tRNA hydrolysates. Digestion of tRNA with ribonuclease T2 generates GmG rather than Gm and G and allows the Gm region of the HPLC profile to be examined in more detail. This region does not contain any dinucleotides arising from the ribonuclease T2 digestion as judged by redigestion with snake venom phosphodiesterase and alkaline phosphatase (data not shown). The HPLC profile of this region is shown in Fig. 4. In addition to Q, m’G, and ac4C, the peaks designated XI , X2, and X3 are present.

above panel.

each com-

Since all of the nucleosides identified in the tRNA hydrolysate were shown to be stable under the digestion conditions used, it is unlikely that Xi, XZ, and X3 correspond to nucleoside breakdown products. Therefore, X 1, X2, and X3 may be uncharacterized intermediates in the biosynthesis of those nucleosides identified in Fig. 3. Alternatively, they may correspond to modified nucleosides found in sequenced tRNAs which are not yet structurally characterized (23). The level of detection of nucleosides is such that only 1 &,-, unit of unfractionated tRNA is required to detect all of the major and minor modified nucleosides reported to be present in S. typhimurium and E. coli tRNA. It is estimated that if other modified nucleosides were present, they would be found only in very minor species of tRNA, present at the level reported for the minor tRNA”= in E. coli

COMPLETE

ANALYSIS

OF tRNA-MODIFIED

Time

NUCLEOSIDES

(min)

FIG. 3. Reverse-phase separation of nucleosides from S. typhimurium tRNA. One Azm unit of bulk tRNA hydrolysate was chromatographed and the absorbance at 254 nm plotted. U* has been tentatively identified as cmnmWJ (28).

(26). Using higher levels of tRNA (4 AZ60units) we were unable to detect any of the other known modified nucleoside listed in Fig. 2 which are not listed in Table 1 as components of S. typhimurium tRNA. Therefore, if present, these nucleosides would occur with a frequency of not more than 0.1 mol per 100 mol of pseudouridine, that is, at about one-twentieth the level of mt6A. They would therefore be unique to very minor tRNA species. DISCUSSION

Reverse-phase high-performance liquid chromatography is a powerful analytical technique suitable for the separation of molecules differing widely in their polarity and has therefore gained wide popularity as a rapid method for analyzing complex biological samples (25,29). Previous work from this laboratory has used HPLC to examine the nu-

cleotide and small molecule content of bacterial cells (25). Factors affecting nucleoside chromatography on reverse-phase columns have been studied in detail (12) and an appraisal of the method for analyzing tRNA hydrolysates has been made ( 13). We have used gradient elution to resolve the strongly hydrophobic nucleosides and ammonium acetate as the low-strength eluant in order that compounds of interest can be recovered by lyophilization for further characterization (25). Using this procedure, nucleosides are eluted essentially in the order shown by Gehrke and workers (13) with the notable exception of m’G, which elutes after G rather than before. This can be attributed to the slightly higher pH of our elution buffer and the highly pHdependent retention characteristics of this compound on reverse-phase columns ( 12). As expected, increasingly hydrophobic molecules elute later as depicted in the series A,

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TABLE 1 NIJCLEOSIDE

Nucleoside C G

A U gOI m5U SW m’G m*A Cm m’G mcmo5u”“’

Cm mnm5s2U cmo5U ms2i06A s*c acp’U m6A Urn

Q tbA mtbA cmnm5s2U’“’ ms2i6A i6A a& m’A m*G

COMPOSITION

OF S. typhimurium

(a) Mole of nucleoside per mole of pseudouridine X 100 1488 1473 953 793 100 59 44 42 12.51 11.74 11.50 7.0 1 6.96 6.92 6.47 5.48 4.94 4.51 4.04 3.56 3.00 2.41 2.17 1.92 1.83 1.66 1.45 1.13 0.96 0.25

(128) (90) (94) (25) (-) (3) (3) (2) (0.14)

(1.W (0.8 1) (1.55) (0.94) (0.17) (1.53) (2.09) (0.68) (0.49) (0.89) (0.26) (0.53) (0.64) (0.23) (0.45) (0.52) (0.17) (0.49) (0.12) (0.76) (0.11)

tRNA

(6) 96 98 98 97 91 96 97 97 97 < 17’” 280’” 89 10 89 87 97 92 93 94 92 12 96 92 95 86 97 98 91 97 97

(4

1344 1412 896 649 100 59 37 42 15 12 4 8 9 5 16 4 4

-

10 13 3 6 -

100 55-62 30-43 20-25 5-13 5 7-8 3 5 2 7-8 5-7 3 3.5-l 1

2.5-4 0.15-0.5 4 2-17

(a) The nucleoside composition was calculated relative to pseudouridine. 1 A 2W unit of unfractionated tRNA was estimated to contain 2.66 nmol of pseudouridine (SD = 0.16). Data shown were determined from five individual tRNA preparations. Numbers in parentheses are standard deviations. The third column is the percentage of nucleoside retained by the phenyl boronate affinity column. (i) S. typhimurium hisT mutants, lacking pseudouridylate synthase I (32), were shown to have 36% less pseudouridine in their tRNA than wild type 5’. typhimurium. The levels of all other modified nucleosides remain unchanged. (ii) Data from RNAse T2 digests and boronate chromatography of complete tRNA hydrolysates containing the ribose-methylated nucleosides. (iii) Estimated using the integrated absorption data for cmo?J. By HPLC, 1 A254 unit of mcmo5U yielded exactly 1 A 254unit of cmo%J following alkaline hydrolysis. (iv) Estimated using the integrated absorption data for mnm%*U. ’ Data derived from Ref. (26). Levels of A,G,C,U were calculated assuming tRNAP” and tRNACy5 were present at the same level as tRNATy’. tRNAPm was assumed to have an average A,G,C,U composition. Modified nucleoside composition of bulk tRNA was computed from relative tRNA abundances and the known nucleoside content of individual tRNA species (23). ‘Data from Ref. (21).

ms2A, io6A, ms2i06A, i6A, ms2i6A. Interestingly, the carbamoyl threonine side chain of t6A also behaves as a hydrophobic addition

to the adenosine molecule rather than increasing its polarity, a property which might not have been predicted from the knowledge

COMPLETE

Time

ANALYSIS

OF tRNA-MODIFIED

(min)

FIG. 4. As Fig. 3, but tRNA was digested with ribonuclease Tz. The region corresponding to Q-mcmosU is shown.

of the net negative charge carried by the free carboxylic acid function of the side chain of t6A (20). However, ms2t6A eluted ahead of ms*A rather than later as would be predicted from the elution sequence A, t6A, m6A, mt6A, ms*A. The reason for this is unclear. Certain post-transcriptional modifications alter the pK of the parent nucleoside and introduce an electrostatic charge on the ring (19). This effect explains why m’A behaves as if it were a polar molecule with respect to A and m6A, and could also account for the altered mobility of m$t6A with respect to ms*A. We have found it unnecessary to thermostat the column to obtain reproducible chromatographic separations; the range of temperatures experienced in our laboratory do not change the relative elution positions of any of the nucleosides resolved in our separation. Absolute retention times show little variation under ambient run conditions. Over a period of 1

NUCLEOSIDES

11

year with three different columns the retention time of pseudouridine varied by 0.2 min, m5U by 0.5 min, A by 0.8 min, and ms2i6A by 1.2 min. The chromatographic method used has enabled a detailed analysis of the modified nucleoside composition of S. typhimurium tRNA to be made. Digestion of pure tRNAs of known base composition and careful analysis of S. typhimurium tRNA hydrolysates has shown the procedures used to be accurate and free of artifacts.4 All 24 modified nucleosides previously reported for E. coli and S. typhimurium tRNAs, with the exception of dihydrouridine which does not absorb at 254 nm, have been identified and are present in the expected amounts (2 1,26,27). The observed nucleoside composition of total S. typhimurium tRNA (Table 1) is a weighted average, dependent upon the nucleoside composition of each isoacceptor tRNA and its relative abundance. Differences in the abundance of individual tRNAs are likely to account for most of the differences between the experimental values reported here for S. typhimurium and those derived from Ref. (26) for the nucleoside composition of E. coli tRNA. Since both t6A and acp3U were shown to be stable under the digestion conditions employed, the rather low levels of these nucleosides found in S. typhimurium tRNA hydrolysates is therefore likely to be a reflection of isoaccep tor tRNA abundance. It is noteworthy that both cmo’U and mcmo’U are detected, a finding confirmed by the absence of these two modifications from S. typhimurium aroA mutants (( 18); M. Buck, B. N. Ames, unpublished observations). Whether mcmo5U has not been detected in sequenced tRNAs because of its instability in alkali or because it is present only in, as yet, unsequenced tRNAs remains to be seen. The unusual relationship between mcmo5U, cmo’U, and the aromatic biosynthetic pathway has been reported previously ( 18). The characterization of ms2i06A 4 Recently a combination of nuclease P, and bacterial alkaline phosphatase has been used successfully to hydrolyze tRNA for HPLC analysis (36).

12

BUCK, CONNICK,

and cmnm%‘U from S. typhimurium tRNA is reported in detail elsewhere (28). The three unidentified peaks (X, , X2, X,) in the HPLC run are present at fairly low levels as judged by their 254~nm absorbance, and may therefore represent biosynthetic intermediates or very rare nucleosides which are found only in minor tRNA isoaccepting species. Although 1 A260 unit of unfractionated tRNA hydrolysate is routinely analyzed and allows the detection of the rare nucleosides mt6A and ac4C present at very low molarity in the unfractionated tRNA, chromatography of as little as 0.1-0.2 AZ60unit of a pure tRNA isoaccepting species is sufficient to determine its modified nucleoside composition. Advances in detector sensitivity are likely to substantially increase the sensitivity of the method and permit the analysis of tRNA which is available in only very limited amounts from organelles. Additionally, the nondestructive nature of the procedure allows the recovery of nucleosides for further characterization. Postlabeling using the trialcohol procedure of Randerath (2) could easily be applied to assess the presence of very rare nucleosides in recovered column fractions in the absence of the large excess of other nucleosides. The technique described is suited to studying the loss of post-transcriptional modifications through mutation; hi.sT mutants (32) have been shown to lack appreciable amounts of pseudouridine from their tRNA (Table 1) and S. typhimurium aroA mutants to lack cmo5U and mcmo’U (see above). Although the modified nucleoside composition reported by us is for S. typhimurium in a balanced state of growth, alterations in the modified nucleoside composition of tRNA may play an important role in integrating and regulating the metabolism of the bacterial cell (30-35). We are currently examining this proposal using the HPLC methodology described in this paper and have found a number of changes in the post-transcriptional modification of tRNA which are associated with certain physiological stresses (see Refs. (34,35)). This will be the subject of a separate com-

AND AMES

munication. The technique described is of course equally suited to analyzing tRNA from higher organisms, and will be useful in determining which modified nucleosides are involved in the changed distribution of isoacceptor tRNAs seen in differentiating tissues and tumor cells (1,30). ACKNOWLEDGMENTS We thank Barry Bochner, Rick Cathcart, Harold Kammen, and Dorothy Maron for their help and advice. M. Buck was recipient of American Cancer Society Postdoctoral Fellowship J-5-82. This work was supported by NIH Grant GM-19993 to B.N.A. REFERENCES 1. Nishimura, S. (1979) in Transfer RNA: Structure, Properties, and Recognition (Schimmel, P. R., Soil, D., and Abelson, J. N., eds.), Monograph 9A, pp. 59-79, Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y. 2. Randemth, E., Yu, C. T., and Randerath, K. (1972) Anal. B&hem. 48, 172-198. 3. Silberklang, M., Gillum, A. M., and RajBhandary, U. L. (1979) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 59, pp. Xl109, Academic Press, New York. 4. Randerath, K., Gupta, R. C., and Randerath, E. (1980) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 65, pp. 638-680, Academic Press, New York. 5. Rogg, H., Brambilla, R., Keith, G., and Staehelin, M. (1976) Nucl. Acids Res. 3, 285-295. 6. Nishimura, S. (1979) in Transfer RNA: Structure, Properties, and Recognition (Schimmel, P. R., Soll, D., and Abelson, J. N., eds.), Monograph 9A, pp. 547-552, Cold Spring Harbor Laboratories, Cold Spring Harbor, N. Y. 7. Feldmann, H., and Falter, H. (197 1) Eur. J. Biuchem. 18, 573-581. 8. Bochner, B. R., and Ames, B. N. (1982) J. Biol. Chem. 257,9759-9769. 9. Brownlee, G. G. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S., and Work, E., eds.), Vol. 3, pp. l-265, NorthHolland, Amsterdam. 10. Gupta, R. C., Randerath, E., and Randemth, K. (1976) Nucl. Acids Rex 3, 2915-2921. 11. Hartwick, R. A., and Brown, P. R. (1976) J. Chromatogr. 126, 679-69 1. 12. Gehrke, C. W., Kuo, K. C., and Zumwalt, R. W. (1980) J. Chromatogr. 188, 129-147.

COMPLETE

ANALYSIS

OF tRNA-MODIFIED

13. Davis, G. E., Gehrke, C. W., Kuo, K. C., and Agris, P. F. (1979) J. Chromatogr. 173, 281-298. 14. Agris. P. F., Tompson, J. G., Gehrke. C. W., Kuo. K. C., and Rice. R. H. (1980) J. C/rroma(ogr. 194, 205-212.

15. Watanabe, K., Oshima, T., Iijima, K., Yamaizumi, Z., and Nishimura, S. (1980) J. Biochem. 87, l18. 16. Alper, M. D., and Ames, B. N. (1978) J. Bacferiol. 133, 149-157. 17. Avital, S., and Elson, D. (1969) B&him. Biophys. Acta 179, 297-307. 18. Bjork, G. R. (198O)J. Mol. Biol. 140, 391-410. 19. Dunn, D. B., and Hall, R. H. (1975) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., pp. 65-215, CRC Press, Boca Raton, Fla. 20. Hall, R. H. (197 1) The Modified Nucleosides in Nucleic Acids, Columbia Univ. Press, New York/ London. 21. Hall, R. H., and Dunn, D. B. (1975) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., ed.), 3rd ed., pp. 2 16-250, CRC Press, Boca Raton, FIa. 22. Macon, J. B., and Wolfenden, R. (1968) Biochemistry I, 3453-3458. 23. Sprinzl. M.. and Gauss. D. H. ( 1982) Nrrcl. .lci& Rex IO, rl-r55.

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24. Hageman, J. H., and Kuehn, G. D. (1977) Anal. Biochem. SO, 547-554. 25. Payne, S. M., and Ames, B. N. (1982) Anal. Biochem. 123, 151-161. 26. Ikemura, T. (1981) J. Mol. Biol. 146, I-21. 27. Cimino, F., Traboni, C., Colonna, A., Izzo, P., and Salvatore, F. (1981) Mol. Cell. Biochem. 36, 95104. 28. Buck, M.. McCloskey, J. A.. Basile, B., and Ames. B. N. (1982) NacI. Acids Rev. 10, 5649-5662. 29. Brown, P. R., and Krstulovic, A. M. (1979) Anal. Biochem. 99, l-2 1. 30. Littauer. U. Z.. and Inouye, H. (1973) Ann. Rev. Biochem. 42, 439-470. 3 1. Void. B. S.. (1978) J. Bucteriol. 135, 124-I 32. 32. Singer, C. E., Smith, G. R., Cortese, R., and Ames, B. N. (1972) Nature (London) New Biol. 238, 7274. 33.

Yanofsky, C., and Soll. L. (1977) J. Mol. Biol. 113, 663-677.

McLennan, B. D., Buck, M., Humphreys, J., and Griffiths, E. (198 1) Nucl. Acids Rex 9,2629-2640. 35. Tumbough, C. L., Neill, R. J., Landsberg, R., and Ames, B. N. (1979) J. Biol. Chem. 254, 5 11 l5119. 36. Gehrke. C. W., Kuo, K. C., McCune. R. A., Gerhardt, K. 0.. and Agris, P. F. (1982) J. Chromafogr. 230, 297-308. 34.